Study on cancer stem cell labeling and inhibition efficiency of LV3 nanocomplex in vitro

Life ScienceS | Medicine Vietnam Journal of Science, Technology and Engineering 47march 2021 • Volume 63 Number 1 Introduction Despite abundant ongoing research efforts, cancer remains one of the most challenging diseases to treat globally. Due to the heterogeneous nature of cancer, one of the major clinical challenges is the ability of cancer to develop resistance in therapeutic development. It has been hypothesized that cancer stem cells (CSCs) are the cause of this resistance and targeting their treatment will lead to tumour regression [1]. CSCs accounts for a small percentage of tumours and can regenerate into various tumorous cell types causing the growth and expansion of malignancy. CSCs present drug-resistant abilities and overcome radiotherapy. Then, the survival of cancer stem cells after treatment allows the tumour to recur and spread throughout the body. Therefore, CSCs are considered a promising target for research and discovery of more effective anticancer drugs or therapies. CSCs are characterized by several specific surface markers. A pentaspan transmembrane glycoprotein, CD133, has been suggested to mark cancer stem cells in various tumour types. However, the accuracy of CD133 as a cancer stem cell biomarker has been highly controversial [1]. CD133 is known as prominin-1, a transmembrane glycoprotein, and is a common surface marker for CSCs, which are inside of various cancer tumours. This transmembrane CD133 glycoprotein includes an extracellular N-terminus and an intracellular C-terminus, which have been used as an efficacious typical surface antigen to detect and to isolate CSCs [2]. As recognized, traditionally nanotechnological biomedicine heighten pharmaceutical properties and reduce the systemic toxicity of chemotherapy through selectively targeting and effectively transferring anticancer drugs to tumours. Nanoparticles usually improve the therapeutic index of the chemotherapeutic drugs that are enveloped inside or combined with the nanoparticle surfaces. For Study on cancer stem cell labeling and inhibition efficiency of LV3 nanocomplex in vitro Le Nhat Minh1, Vo Trong Nhan1, Thi Thao Do2, Tran Thu Huong3, Le Tri Vien4, Phung Thi Kim Hue1* 1Hung Vuong Gifted High School, Gia Lai, Vietnam 2Institute of Biotechnology, Vietnam Academy of Science and Technology, Vietnam 3Institute of Materials Science, Vietnam Academy of Science and Technology, Vietnam 4Institute of Health Research and Educational Development in Central Highlands, Vietnam Received 2 March 2020; accepted 22 May 2020 *Corresponding author: Email: whitelily109@gmail.com Abstract: Cancer stem cells (CSCs) are the cancer cells that have abilities to self-renew, differentiate into defined progenies, and to initiate and maintain tumour growth. They also contribute to cancer metastasis and therapeutic resistance, both of which are the principal causes of cancer mortality. Therefore, finding efficient fluorescent materials for CSC labelling and basic research is an urgent need. Thus, this research is focused on using a rare-earth element, which is the fluorescent Tb3+ nano-ion, and the CD133 monoclonal antibody to create a CSC-targeting nanocomplex (LV3). Tb3+ nanorods were silica-surface treated and -NH2 activated before being conjugated with the monoclonal antibody (mAb) against CD133, a typical CSC surface marker. The use of robust fluorescent Tb3+ nanorods was to decrease the toxicity of a high-dose prober while the CD133 mAb would increase the CSC’s specific binding capacity of the LV3. The fluorescent properties of the coupled LV3 complex were measured and CSC-targeting label activities on the pluripotent human embryonal carcinoma cell line (NTERA-2) were observed. The obtained results presented fluorescent images of LV3 exposed to NTERA-2 cells under microscopy. LV3 also demonstrated that it effectively labelled up to 99.68% of the tested NTERA-2 cells. By contrast, LV3 only labelled 1.44% of the CCD-18Co human healthy cells. On the other hand, LV3 exhibited anti-CSC activity, which inhibited 11.14% in vitro and 30.5% tumourspheroid growth of NTERA-2 cells. In conclusion, LV3 showed its efficiency in specific CSC target labelling and inhibition, which could be further applied to fundamental and preclinical research. Keywords: cancer stem cells, CCD-18Co, CD133 monoclonal antibody, ion Tb3+, LV3, NTERA-2. Classification number: 3.2 DOI: 10.31276/VJSTE.63(1).47-53 Life ScienceS | Medicine Vietnam Journal of Science, Technology and Engineering48 march 2021 • Volume 63 Number 1 example, rare-earth elements such as Tb3+ nano-materials or cation terbium (III) emitting green fluorescence would effectively assist in detection and treatment [3]. Thus, experimental and clinical applications of CSC labelling and tracking are interesting to evaluate cell location. Rare-earth- based nanotechnology would be very helpful [4]. Also, several studies on nanomaterial labelling effects targeting cancer cells have been reported elsewhere [5, 6]. According to the previous report from the authors in this study [7], Tb3+ a rare-earth ion could constitute a fluorescent nanomaterial as terbium orthophosphate monohydrate (TbPO4.H2O) in the form of hexagonal crystal structure. Fluorescent spectra of TbPO4.H2O nanomaterials at pH=2, incubated at 200oC for 24 h, coated by silica, and functionalized with -NH2 measured at 355 nm was determined by iHR55 system (Jobin-Yvon). The fluorescent images of human colorectal adenocarcinoma cells (HT-29) were observed by the effects of the complex of the surface-functionalized TbPO4.H2O- NH2 and the anti-CD133 mAb [7]. Our previous study also reported that the fluorescent Tb3+ nano-ion and CD133 mAb conjugation (ET complex) gave marks on 26.89% (of colorectal cancer cells) [8] and 97.74% (of NTERA-2 cells) compared to the control, respectively [9]. To continue this study, we modify the content of one component of the RT2 [9] complex to make LV3 and attempted to evaluate the CSC growth inhibition activities of the LV3 complex. Material and method Materials LV3 is made from nano Tb3+-monoclonal antibody (RT) transport system [9]. The pluripotent human embryonal carcinoma cell line (NTERA-2) and the human healthy cell line (CCD-18Co) were kindly provided by Dr. P. Wongtrakoongate, Mahidol University, Thailand, and Prof. Chi-Ying Huang, National Yang-Ming University, Taiwan. Cells were maintained in DMEM medium supplement with 10% foetal bovine serum and 1% antibiotics (antibiotics-antimycotics solution, Invitrogen, Carlsbad, CA, USA) in a humidified incubator with 5% CO2 at 37oC. Cultured medium so-called Dulbecco’s Modified Eagle Medium (DMEM), Foetal bovine serum (FBS), Trypsin-EDTA, antibiotics (antibiotics-antimycotics) were purchased from Invitrogen (Carlsbad, CA, USA). Human CD133 monoclonal antibody and human CD133 antibody conjugated with FITC (FITC-CD133) were from Miltenyi Biotec (Bergisch Gladbach, Germany). Other chemicals were provided by Sigma Aldrich (St. Louis, MO, USA). In vitro cell culture The in vitro cell culture was carried out by following the protocols from ATCC Cell Bank (American Type Culture Collection, USA). Accordingly, NTERA-2 and CCD-18Co cells were cultured in T75 flask with DMEM supplemented by 2 mM L-glutamine, 10% foetal bovine serum (FBS), and 1% antibiotic (Anti-Anti solution). The cells were subcultured every 3-5 d with a ratio of 1:3 and incubated in humid conditions of 37oC and 5% CO2. Labelling cells with LV3 - Cancer stem cells and healthy cells imaging: using LV3: NTERA-2 and CCD-18Co, cells were pre-seeded into a 96-well plate at 10,000 cells/well and incubated at 37°C, 5% CO2 for 24 h. Then, the culture medium was replaced with 10% formaldehyde to fix the cells for 10 min at room temperature. The cells were triple rinsed with phosphate buffered saline (PBS) to thoroughly remove formaldehyde. Then, 10 µl of LV3 in 190 µl of PBS were placed into each well and incubated at 4°C for 1 h. The unbound sample was removed and triple rinsed with PBS. Finally, 100 µl PBS was added to the wells before observation using an Olympus Scan^R fluorescence microscope (Olympus Europa SE & Co.KG, Hamburg, DE). - Determining the number of marked cells (through CD133 surface marker) by flow-cytometry: cancer stem cells (NTERA-2) and healthy cells (CCD-18Co) were seeded into a 6-well plate and incubated at 37oC, 5% CO2 overnight. After 24 h of incubation, the cells were harvested with trypsin-EDTA and collected into a falcon tube. Cells were re-suspended with DMEM medium containing 2% FBS, LV3, or anti-CD133-FITC mAb and then incubated at 4°C for 10-15 min while protected from light. The number of labelled cells (out of 10000-12000 counting cells) were measured and analysed by Novocyte flow cytometry system (ACEA Bioscience Inc.) and NovoExpress software. LV3 cytotoxic determination The MTT assay was employed according to Mosmann (1983) [10] to measure the cytotoxic activity of the LV3 nanocomplex. In short, cells were seeded in 96-well plates and triplicated, then treated with LV3 at various concentrations for 72 h at 37oC, 5% CO2. Then, 10 µl MTT (5 mg/ml) was added to each well and incubated at Life ScienceS | Medicine Vietnam Journal of Science, Technology and Engineering 49march 2021 • Volume 63 Number 1 37oC for 4 h. The medium was discarded and the formazan crystal was dissolved by using 50 µl/well dimethlysulfoxide (DMSO). The OD values were measured at 540 nm by a spectrophotometer (BioTek, ELx800). The number of survived cells was calculated by the formula: 3 bovine serum (FBS), Trypsin-EDTA, antibiotics (antibiotics-antimycotics) were purchased from Invitrogen (Carlsbad, CA, USA). Human CD133 monoclonal antibody and human CD133 antibody conjugated with FITC (FITC-CD133) were from Miltenyi Biotec (Bergisch Gladbach, Germany). Other chemicals were provided by Sigma Aldrich (St. Louis, MO, USA). In vitro cell culture The in vitro cell culture was carried out by following the protocols from ATCC Cell Bank (American Type Culture Collection, USA). Accordingly, NTERA-2 and CCD-18Co cells were cultured in T75 flask with DMEM supplemented by 2 mM L- glutamine, 10% foetal bovine serum (FBS), and 1% antibiotic (Anti-Anti solution). The cells were subcultured every 3-5 d with a ratio of 1:3 and incubated in humid conditions of 37oC and 5% CO2. Labelling cells with LV3 - Cancer stem cells and healthy cells imaging: using LV3: NTERA-2 and CCD- 18Co, cells were pre-seeded into a 96-well plate at 10,000 cells/well and incubated at 37°C, 5% CO2 for 24 h. Then, the culture medium was replaced with 10% formaldehyde to fix the cells for 10 min at room temperature. The cells were triple rinsed with phosphate buffered saline (PBS) to thoroughly remove formaldehyde. Then, 10 µl of LV3 in 190 µl of PBS were placed into each well and incubated at 4°C for 1 h. The unbound sample was removed and triple rinsed with PBS. Finally, 100 µl PBS was added to the wells before observation using an Olympus Scan^R fluorescence microscope (Olympus Europa SE & Co.KG, Hamburg, DE). - Determining the number of marked cells (through CD133 surface marker) by flow-cytometry: cancer stem cells (NTERA-2) and healthy cells (CCD-18Co) were seeded into a 6-well plate and incubated at 37oC, 5% CO2 overnight. After 24 h of incubation, the cells were harvested with trypsin-EDTA and collected into a falcon tube. Cells were re-suspended with DMEM medium containing 2% FBS, LV3, or anti- CD133-FITC mAb and then incubated at 4°C for 10-15 min while protected from light. The number of labelled cells (out of 10000-12000 counting cells) were measured and analysed by Novocyte flow cytometry system (ACEA Bioscience Inc.) and NovoExpress software. LV3 cytotoxic determination The MTT assay was employed according to Mosmann (1983) [10] to measure the cytot xic activity of the LV3 nanocomplex. In short, cells were seeded in 96-well plates and triplicated, then treated with LV3 at various concentrations for 72 h at 37oC, 5% CO2. Then, 10 µl MTT (5 mg/ml) was added to each well and incubated at 37oC for 4 h. The medium was discarded and the formazan crystal was dissolved by using 50 µl/well dimethlysulfoxide (DMSO). The OD values were measured at 540 nm by a spectrophotometer (BioTek, ELx800). The number of survived cells was calculated by the formula: % survived = ( ) ( ) ( ) ( ) . Measurement of 3D tumoursphere growth inhibitive activities BALB/c mice macrophages were isolated using a Macrophage Mouse Isolation Kit (Peritoneum) (Miltenyi Biotech., Bergisch Gladbach, Germany). The isolated cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics at 37oC and 5% CO2. Measure ent of 3D tumoursphere growth inhibitive activities BALB/c mice macrophages were isolated using a Macrophage Mouse Isolation Kit (Peritoneum) (Miltenyi Biotech., Bergisch Gladbach, Germany). The isolated cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics at 37oC and 5% CO2. In order to form tumour spheroids, the hanging drop method was performed. The 1500 NTERA-2 cells in 20 µl of medium were dropped onto the bottom of the 60 mm tissue culture dish lid before inverting that lid onto the 5-ml medium filled bottom dish. The dish was then incubated at 37°C, 5% CO2, and 95% humidity. After 3-d incubation, cell aggregates formed. These 3D tumourspheres were further co-cultured with macrophages in a 96-well plate. Wells were covered with 1% agarose before the spheroids were transferred to the wells. The macrophage cells were then co-cultured with the spheroids in the wells. The LV3 treatment was performed by directly adding LV3 into the co-culture wells and further incubated for 3 d. The growth of the spheroids was observed under microscopy. The images were analysed using ImageJ software. Statistical analysis The data was reported as mean±standard deviation (SD) and analysed by the GraphPad Prism 7 software using an unpaired t-test. A p<0.05 was considered statistically significant. Results and discussion Probing NTERA-2 and CCD-18Co cells and with LV3 fluorescent nanocomplex The results exhibited that the NTERA-2 cells were labelled by LV3 and displayed strong fluorescence under fluorescence microscopy (Fig. 1, Table 1). The healthy cells known as CCD-18Co did not emit any corresponding signal under the same condition (Fig. 2). Therefore, LV3 could be specifically targeting cancer stem cells. The obtained results were consistent with the research of Le Nhat Minh, et al. (2019) [9]. Fig. 1. NTERa-2 cells was probed after 1 h of incubation either with LV3, TbPO4.H2O.silica-NH2, CD133-FITC, or without LV3 (negative control) by fluorescence microscopy on an Olympus Scan^R. LV3 TbPO4.H2O.silica-NH2 CD133-FITC Negative control Life ScienceS | Medicine Vietnam Journal of Science, Technology and Engineering50 march 2021 • Volume 63 Number 1 Table 1. Labelling performance of cancer stem cells and healthy cells. Samples The number of fluorescent detected cells (%) Cancer stem cells (NTERA-2) Healthy cells (CCD-18Co) LV3 99.68±3.85 1.44±0.11 TbPO4.H2O.silica-NH2 0.57±0.07 0.31±0.02 CD133-FITC 95.83±7.31 1.17±0.06 Negative control 0.10±0.02 0.20±0.04 Table 2. Total intensity of samples in examined cell lines. Samples Total intensity NTERA-2 CCD-18Co LV3 6011±62.62** 0 CD133-FITC 5497±42.87 0 Negative control 19.00±3.07 0 Negative control sample; lV3 - experimental sample. Data is expressed as mean ± Se (n=3) combined from three repeated experiments. *Significant differences (t-test, p≤0.05) and **(p≤0.01). The fluorescent intensity was analysed in the LV3- treated NTERA-2 cell line by fluorescence microscope as shown in Table 2. By fluorescence spectroscopy, NTERA-2 emits fluorescence intensity at 6011±62.62 FU, which is statistically significant compared to the negative control. Meanwhile, fluorescence intensity in healthy cells were not measurable. This result is consistent with the study of Le Nhat Minh, et al. (2019) [9] and is demonstrated in Fig. 1. LV3 fluorescent labelling performance by flowcytometry evaluation We used flow-cytometry to evaluate the labelling specificity of LV3. Detailed results are shown in Table 1 and Fig. 3. The results showed that the LV3 probed 99.68% of NTERA-2 cells, which was higher than the respective number (0.10%) of the negative control. As a result, it is seen that LV3 labelled NTERA-2 cells more efficiently than the RT labelling of the colorectal cancer cell [9]. Further research is required to elucidate how LV3 could label a cancer stem cell better than in other cancer cell lines. Besides, LV3 could not label and distinguish CCD-18Co. Thus, LV3 is an effective material for labelling CSCs. These preliminary results demand more studies in vivo and clinical testing. Recent clinical studies have shown that high expression of CD133 in tumours plays an important role as a prognostic marker of disease progression. As such, a spectrum of immunotherapeutic strategies has been developed to target these CD133 positive cells with the goal of translation into the clinic. In one report, the researcher Mi Y used salinomycin-loaded poly (lactic-co-glycolic acid) - poly (ethylene glycol) nanoparticles conjugated with CD133 antibodies (CD133-SAL-NP) to eliminate CD133+ ovarian LV3 TbPO4.H2O.silica-NH2 CD133-FITC Negative control Fig. 2. CCD-18Co cells was probed after 1 h of incubation either with LV3, TbPO4.H2O.silica-NH2, CD133-FITC, or without LV3 (negative control) by fluorescence microscopy on an Olympus Scan^R. Life ScienceS | Medicine Vietnam Journal of Science, Technology and Engineering 51march 2021 • Volume 63 Number 1 CSCs. The CD133-SAL-NPs efficiently bound to the CD133+ ovarian cancer cells resulting in an increased cytotoxic effect toward CD133+ ovarian cancer cells when compared with the untargeted SAL-NPs and salinomycin. The CD133-SAL-NPs reduced the percentage of CD133+ ovarian CSCs in ovarian cells more effectively than treatment with salinomycin or SAL-NPs, suggesting that CD133-SAL-NP targeted CD133+ ovarian CSCs [11]. Herein, rare-earth nano-ion Tb3+ conjugated with anti- CD133 mAb to formulate LV3 presents promising CSC labelling and specific targeting capacities. This might also be the first developing stage of rare-earth-based nanomaterials for valuable applications in cancer diagnostics and treatment. (a) LV3 NTERa-2 Healthy cells (CCD-18Co) (b) TbPO4.H2O.silica-NH2 NTERa-2 Healthy cells (CCD-18Co) (C) CD133-FITC NTERa-2 Healthy cells (CCD-18Co) (D) Negative control NTERa-2 Healthy cells (CCD-18Co) Fig. 3. Flow-cytometry analysis to determine the number of fluorescent labelled cells with various materials: (a) LV3, (b) TbPO4.H2O.silica-NH2, (C) CD133-FITC, and (D) negative control. Life ScienceS | Medicine Vietnam Journal of Science, Technology and Engineering52 march 2021 • Volume 63 Number 1 Effect of LV3 on the proliferation of NTERA-2 The proliferation of LV3-treated NTERA-2 cell was assessed by using MTT assays. LV3 showed the ability to inhibit the growth of NTERA-2 cells up to 11.14% at a concentration of 10 µg/ml (Table 3). The anti-proliferation of LV3 on NTERA-2 cells was slightly higher than that on CCD-18Co cells. Table 3. The proliferation of complex on NTERa-2 cells and CCD-18Co cells. Samples % proliferation NTERA-2 CCD-18Co LV3 88.86±2.13 95.05±0.68 Negative control 100 100 Effect of LV3 on NTERA-2 spheroids co-culture with macrophages Although several CSCs markers have been reported, one of the most promising and possibly least ubiquitous is CD133, a frequently expressed surface marker on CSCs. Some evidence has indicated that directly targeting CD133 with biological drugs might eliminate CSCs effectively [12]. Table 4. 3D tumour spheroids inhibited under the treatment of LV3. Samples 3D tumour spheroids (%) Standard deviation LV3 69.50* 0.81 Negative control 100.00 0.09 Negative control sample; LV3 - Experimental sample. Data is expressed as mean ± SE (n=3) combined from three repeated experiments. * Significant differences (t test, p≤0.05). In this study, the activity of LV3 inhibited 3D-tumor growth in vitro formed by co-culturing 3D NTERA-2 spheroids with macrophages (Table 4; Fig. 4). As a result, the diameters of 3D spheroids dropped to 69.50% in comparison with that of the untreated negative control after a 3-d treatment. Herein, although LV3 slightly inhibited the growth of NTERA-2 cells (11.14%) in vitro, this nanocomplex strongly inhibited the growth of these 3D NTERA-2 spheroids (30.5%) when co-cultured with macrophages. Among the reported markers of the cancer stem cells, CD133 is the most well-known marker for isolating and studying CSCs in different types of cancer. The CD133 high population of cancer cells are not only capable of self- renewal and proliferation but are also highly metastatic and resistant to therapy. Despite limited information on the physiological functions of CD133, many ongoing studies aim to reveal the mechanisms that CD133 utilizes to modulate cancer dissemination and drug resistance (Liou, 2019) [13]. Thus, the role of anti-CD133 antibodies in the LV3 may reduce the function of CD133 and result in the inhibition of CSCs. According to another report that investigates the cytotoxic, radiation dose-enhancing, and radio-sensitizing ability of five rare-earth oxide nanoparticles on the two immortalized mammalian cell lines U-87 MG and Mo59K, a significant cytotoxicity of Nd2O3 and La2O3 was observed in U-87 MG cells. As aforementioned, the component of LV3 is terbium (Tb3+), which is a typical lanthanide with green fluorescence that has the potential for biomedical labelling and imaging. Seemingly, terbium in LV3 is likely to have an inhibitory effect on CSCs. In this study, LV3 was 3D tumour treated with LV3 3D tumour – negative control Fig 4. The 3D tumourspheres at the day 3 under the treatment of LV3 and negative control under the fluorescence microscope system Olympus Scan^R 100X. Life ScienceS | Medicine Vietnam Journal of Science, Technology and Engineering 53march 2021 • Volume 63 Number 1 demonstrated to be a promising target for drug delivery to CSCs and may be useful as an agent to inhibit the growth of cancer by targeting CSCs. LV3 may, therefore, represent a promising approach for the treatment of cancer. Conclusions LV3, which was a combination between the rare-earth- based Tb3+ nanorod and CD133 monoclonal antibody, was assessed for its fluorescent properties and tumoursphere inhibition using cancer stem cells (NTERA-2) and healthy human colon cells (CCD-18Co). The LV3-probed NTERA-2 cells exhibited strong emission under fluorescent microscopic observation. The NTERA-2 labelling efficiency of the LV3 was 99.68% from flow cytometric analysis whereas healthy cells (CCD-18Co) were weakly probed (1.44%). 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